The overall aim of this research program is to develop a single-cell bioreactor system that will automate the technology for culturing single cells and allow for the rapid selection and optimization of media and growth factors for stem cell growth. This system, which will allow cells to be grown in a sterile environment in which temperature, pH, and atmosphere (e.g., humidity and pO2) are regulated, will monitor continuously in situ the behavior of individual cells (e.g., division, differentiation, and motility) in multiple wells in a 96-well plate using a computer-controlled microscope imaging system with pattern recognition software to drive a robotic pipetting assembly to automatically change media in individual wells to respond to changes in cell number (growth) and phenotype (differentiation). We believe that this new technology will: 1) allow the reproducible growth of pluripotent stem cells; 2) allow automation of the study of cell growth, including the development of optimal substrates and growth factors for cell proliferation and differentiation, in a way that is superior in speed and efficiency compared to existing manual techniques; and 3) have widespread applications to many problems in cell biology, toxicology and tissue engineering. PROPOSED COMMERCIAL APPLICATION: The automated bioreactor's first commercial application will be to grow large populations of stem cells, but we believe that the bioreactor can also be used to determine the optimum growth media for many different types of cells, including neurons, myoblasts, endothelial cells and embryonic stem cells.